Summary and Background: Auditory and vestibular systems are required for proper hearing and balance. These systems require sensory hair cells of the inner ear, to sense sound and process vestibular stimuli. To reliably transmit auditory and vestibular information, hair cells use specialized ribbon synapses. Our studies combine genetic, molecular, and imaging-based approaches to identify the structural and functional processes underlying synapse formation and function in hair cells. For our studies we use the zebrafish lateral-line system in order to study hair-cell development and function, in a live, transparent preparation. We have an extremely powerful collection of transgenic zebrafish that label synaptic structures to either assess synaptic morphology or function using genetically encoded fluorescent proteins. We are combining these microscopy-based approaches with CRISPR technology to create mutant zebrafish in order to identify genes required for synapse formation, function and regeneration. With this knowledge we aim to apply our understanding of these processes in order to understand how to properly reform hair cells and synaptic structures when they are loss or damaged after hearing loss. This report summarizes the fourth year for the Section on Sensory Cell Development and Function. Our main focus has been completing research projects initiated at the NIH, and publishing these studies. The lab continues to demonstrate proficiency in several areas: animal husbandry, the creation of mutant and transgenic zebrafish, electrophysiology, and function-based confocal imaging in zebrafish. General accomplishments for this fiscal year include finalizing the installation of our main zebrafish facility in building 35A. In addition, we several ongoing collaborations with other labs at the NIDCD, and the surrounding area. Through these collaborations we will apply our expertise and tools in zebrafish to advance and complement other aspects of auditory research. Projects in the lab: 1) Understanding the molecular components that are vital for hair-cell synapses Determining the dynamics of Ribeye at ribbon synapses Very little is known regarding the dynamic properties of presynaptic ribbons, although it is known that they are composed predominately of the protein Ribeye. This is largely due to challenges that arise from the ribbon size at the diffraction limit of light microscopy (250 nm). To examine ribbon and vesicle dynamics, we took advantage of the larger size of ribbons (1 m) in transgenic zebrafish expressing Rib b-EGFP to label ribbons and examined the in vivo mobility and turnover of Ribeye by monitoring fluorescence recovery after photobleaching (FRAP). Overall, our findings from this work support a model where the ribbon is structurally dynamic, rather than static scaffold for vesicle movement. Our data provides fundamental insight into ribbon dynamics that will help us understand how ribbons encode sensory information. Examine how Ribeye and ribbon size impacts synapse function Previous work has shown that hair-cell ribbon size is correlated with differences in postsynaptic activity. However, additional variability in postsynapse size presents a challenge to determining the specific role of ribbon size in sensory encoding. To address how ribbon size influences hair-cell ribbon-synapse function, we used a transgenic zebrafish line that overexpresses the main component of ribbons, Ribeye and enlarges ribbons. Our study revealed that ribbon enlargement results in increased ribbon-localized calcium signals, yet reduces afferent spontaneous activity and disrupts the timing of stimulus onseta distinct aspect of auditory and vestibular encoding. These observations suggest that varying ribbon size alone can influence sensory encoding, and give further insight into how hair cells transduce signals that cover a wide dynamic range of stimuli. 2) Determine how a sensory stimulus are encoded within an intact sensory system Synaptic integration among populations of sensory hair cells Analysis of auditory transduction among ensembles of sensory cells in the mammalian inner earin vivois challenging due to their location deep within the temporal bone. To overcome this limitation, we used optical indicators to investigate mechanotransduction among collections of sensory hair cells in intact zebrafish. Our imaging reveals a previously undiscovered disconnect between hair-cell mechanosensation and synaptic transmission. We show that suprathreshold mechanical stimuli able to open mechanically-gated channels, are unexpectedly insufficient to evoke vesicle fusion in the majority of hair cells. Overall, this work has important implications for inner-ear recovery after hair-cell loss, where it is important to understand how many hair cells must be regenerated, in order to translate sensory stimuli into meaningful behavior. We are currently investigated the mechanism underlying sensory integration and synaptic facilitation among populations of hair cells in vivo. 3) Determine how synaptic calcium modulate hair-cell synapse assembly Our current understanding of hair-cell synapse formation has been largely obtained by studies that examined morphological or functional changes at single time points during development. Although this work has been informative, development is dynamic, and local synaptic activity is thought to play an active role shaping synapse assembly. We have used the zebrafish model to examine the relationship between synaptic activity and ribbon assembly in vivo. Our previous findings indicated that a pharmacological activation or inhibition of synaptic calcium could push developing synapses towards assembly or disassembly, respectively, on relatively short timescales. Currently we have examined how presynaptic calcium levels are controlled at developing ribbons. Our work indicate that hair-cell synapse assembly is controlled by calcium stores. This work provides mechanistic insight into how activity in hair cells regulates synapse assembly during development and knowledge required to reform synapses under pathological conditions.